The HIV genome is tightly compressed (FIG. 1). At least 30 different RNA transcripts are produced by splicing using the six splice acceptors and two splice donor sequences [see references 85, 86]. The structural proteins encoded by HIV are chemically similar to those of the C-type retroviruses and like them are encoded as polyproteins by the gap (group antigen), pol (polymerase) and env (envelope) genes. Clevage of the polyproteins by the viral protease or cellular enzymes generates eight functional virion proteins. In addition to these structural genes, HIV-1 also caries genes for three regulatory proteins, rev (regulator factor); and two proteins involved in virus maturation, vif (virion infectivity factor) and vpu (viral protein U). The vpr (viral protein R) gene encodes a low copy number virion component. In the closely related viruses HIV-2 and simian immunodeficiency virus (SIV) vpr is replaced by vpx (viral protein X), a unique virion protein.
Transcription of the HIV genome during virus replication shows distinct kinetic phases (see references 53,59,60,79). The initial products of HIV gene expression are short, multiply spliced mRNAs approximately 1.8 to 2.0 kb in length, which encode the trans-acting regulatory proteins tat,rev (and possibly nef). As infection by the virus develops, and the levels of the tat and rev proteins rise in the infected cells, mRNA production shifts progressively towards production of a family of singly-spliced 4.3 kb mRNAs encoding env and other HIV gene products such as vif and vpr. Finally, late in the infection process, production switches to full-length, unspliced, transcripts which act both as the virion RNA and the mRNA for the gag-pol polyprotein.
To achieve this control of gene expression, the HIV virus relies on the interaction of cellular and virus-encoded trans-acting factors with cis-acting viral regulatory sequences (1,3,53). Initiation of transcription relies largely on the presence of binding sites for cellular transcription factors in the viral long terminal repeat (LTR) (28). In contrast, the virally encoded regulatory proteins tat and rev exert their activity via cis-acting sequences encoded within HIV messenger RNAs. The trans-activation-responsive region (TAR) is required for tat activity, and is located in the viral long terminal repeat (LTR) between residues +1 and +79 (5,9,10,11,12,13,14,16,27,38). In rev minus cells only the short spliced transcripts appear in the cytoplasm. It therefore seemed likely that a regulatory sequence was present in one of regions removed form regulatory gene mRNAs by splicing. After a systematic search, a cis-acting sequence required for rev activity, was mapped to a complicated RNA stem-loop structure located within the env rading frame. This sequence has been named the rev-responsive element (RRE). The rev-responsive element (RRE) has been localized to a 234-nucleotide long sequence within the env gene (47,51,54,65,67,68,77). Similar regulatory proteins and target sequences are used by HIV-2 and SIV (8,66). The HTLV-1 virus rex gene product appears to function analogously to rev, and can functionally substitute for rev to promote viral gene expression (76).
The distinct kinetic phases of HIV transcription are now believed to reflect the intracellular levels of the regulatory proteins tat and rev. Initially, binding of host transcription factors to the LTR induced basal level transcription of the early mRNAs including tat. As tat levels rise, increased transcription from the LTR is stimulated by the trans-activation mechanism. This leads to further increases in tat levels, and also stimulates production of rev. Production of the viral sturctural proteins begins once rev levels have risen to sufficiently high levels to promode export of messenger RNAs carrying the rev-responsive element (RRE) sequence. The HIV growth cycle may also include a latent stage where viral gene expression is silent because transcription from the viral LTR produces insufficient amounts of regulatory proteins to initiate the lytic growth cycle.
Becuase the rev protein acts post-transcriptionally to mediate the shift towards expression of the late, largely unspliced viral mRNAs (53,60,78,79), rev protein was initially proposed to be a resulator of splicing in HIV. Subsequent work has shown that expression of rev protein permits the appearance in the cytoplasm of transcripts carrying RRE sequences. In the absence of rev, mRNAs carrying the RRE sequence are retained in the nucleus (52,54,65). Although mRNA presursors carrying heterologous splice donor or acceptor sequences may become rev-responsive by addition of the intact RRE sequences (65), it is still unclear whether the effects of the rev protein are coupled to splicing itself or if a still undefined pathway regulates the export of mRNA from the nucleus. In vitro, the rates of splicing of strong splice donor and acceptor sequences, such as from the globin gene, appear to be insensitive to the presence of RRE sequences, suggesting that rev function competes with the splicing function (46).
Rev recognition of the RRE, like tat recognition of TAR, is due to direct binding [33,34,47,68,73,84]. Binding is tight (K.sub.d =1-3 nM) and highly specific for the RRE [33, 34,84]. However, the binding behaviour of rev to RRE RNA is much more complex than the binding of tat to TAR RNA. As the concentration of rev increases, progressively larger complexes with RRE RNA are formed, whereas tat only forms one-to-one complexes with TAR RNA.
The simplest explanation for the RNA binding behaviour of rev is that the protein binds initially to a high affinity site and that subsequently additional rev molecules occupy lower affinity sites [33]. We have recently mapped the high affinity rev binding site to a purine-rich "bubble" located near the 5' end of the RRE [87]. Mutations that disrupt or delete the "bubble" abolish RRE activity [51,68]. The low affinity binding reaction is the result of both protein-protein and protein-RNA interactions. At high concentrations rev polymerizes and forms long filaments 14 nm wide and up to 1,500 nm long. Because of its ability to polymerize, when rev is mixed with HIV mRNAs the RNA is packaged into rod-like ribonucleoprotein filaments. Filament assembly appears to be nucleated by the binding of rev to the RRE and is much more efficient on RNA molecules carrying a functional RRE sequence than on molecules that do not include an RRE sequence than on molecules that do not inlcude an RRE sequence [87].
The RNA binding properties of rev have led us to propose that rev blocks splicing simply by packaging unspliced RNA transcripts containing the RRE sequence into inaccessible ribonucleoprotein complexes [87]. Although complexes containing rev and viral mRNAs have not yet been isolated from infected cells, there is already indirect evidence in support of this type of mechanism. For example, it is believed that the blocking of splicing in vitro by rev is due to the disruption of spliceosome assembly [46]. Furthermore, the in vivo activity of rev appears to be highly concentration dependent, as would be expected for a mechanism of action based on RNA packaging. Rev-minus viruses can only be rescued by co-transfection with very high levels of rev-expressing plasmids [88].
The packaging model also provides a simple kinetic explanation for the delayed appearance of the virion RNA and physical explanation for how rev can act on RREs placed in a wide variety of positions. During HIV infection high levels of the 4.3 kb mRNAs, such as the env mRNA, are synthesized for several hours before significant levels of the full-length virion RNA is produced [59]. Compared to the 4.3 kb mRNAs, the virion RNA carries additioknal unused splice donor and acceptor towards its 5' end, far away from the RRE where filament formation is suggested to nucleate. If stabilization of the virion RNA requires production of a longer ribonucleoprotein filament than the stabilization of the 4.3 kb mRNAs, it is easy to imagine that this would only take place late in the infectious cycle, when intracellular rev protein concentrations are expected to be maximal.
Although we believe tha the physical properties of rev can alone account for its biological activity, there have been some reports that cellular co-factors(s) are also required [89]. Trono and Baltimore suggested that a human cell contains a species-specific factor required for rev activity after observing that mouse cells infected by HIV have a rev-minus phenotype which can be easily reversed by fusion to human cells [89]. However, it is possible that rev protein levels differed between the various cell lines, and that only sub-threshold levels of rev were expressed in the mouse cells [89]. By contrast, rev is functional in Drosophila melanogaster cells [90].
It is an aim of the present invention to provide an effective method for, and compositions for use in, the inhibition of HIV viral growth within cells, which involves modifying the activity of the regulatory protein rev in the viral growth cycle, and also an assay for screening potential anti-viral agents.